Recombinant bicistronic flaviviruses and methods of use thereof

ABSTRACT

The present invention provides recombinant bicistronic flaviviruses, particularly live attenuated recombinant bicistronic flavivirus, which comprise, in order from 5′ to 3′, a viral 5′UTR, an ORF encoding all viral proteins, an internal ribosome entry site, an exogenous nucleotide sequence that encodes an exogenous polypeptide, and a viral 3′UTR. Infection of a host cell with a recombinant flavivirus provides for expression of the exogenous nucleic acid in a host cell. Such recombinant flavivirus are useful for delivering a protein to a mammalian host; and for eliciting an immune response to the exogenous polypeptide.

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/290,412 filed May 10, 2001, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with U.S. government support under grantnos. R21 AI 44343 and PO1 AI 46007 awarded by the National Institutes ofHealth. The U.S. government may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] The invention relates generally to the field of recombinantviruses and induction of specific immunity.

BACKGROUND OF THE INVENTION

[0004] Yellow fever virus (YFV) 17D is a live, attenuated vaccine thathas been used in humans for over 60 years. More than 300 million peoplehave received the vaccine, with an outstanding record of safety andefficacy. After a single dose, neutralizing antibodies appear in nearly100% of vaccines within 10 days. Immunity is extremely durable, and maybe life long. YFV-17D is very immunogenic in humans, with a 90%immunizing dose of only 5-20 plaque-forming units of virus.

[0005] Yellow fever virus is the prototype member of a family ofviruses, the flavivirus. Virions are spherical and about 40-50 nm indiameter. The nucleocapsid has icosahedral symmetry, contains the RNAgenome, and a single core protein, and is surrounded by a lipid bilayerenvelope. The viral genome is composed of a single-stranded RNA ofpositive polarity. The viral genome contains a single, long open readingframe that encodes 10 viral proteins.

[0006] Flaviviruses have been developed as vehicles for delivery offoreign proteins to a mammalian host for the purpose of generating animmune response to the foreign protein(s). Bonaldo et al. (2000) Mem.Inst. Oswaldo Cruz 95:215-223. In recombinant flaviviruses described todate, a nucleic acid molecule encoding the foreign protein is insertedinto a region encoding viral proteins. Such recombinant viruses encode apolyprotein precursor in which the foreign protein is inserted, andwhich must then be cleaved out. A potential drawback to such recombinantviruses is that, because the polyprotein precursor includes an exogenouspolypeptide, the viral proteins may be misfolded and therefore may notfunction normally.

[0007] There is a need in the art for improved recombinant viruses thatare useful in delivering foreign proteins to a mammalian host. Thepresent invention addresses this need.

LITERATURE

[0008] WO 93/11250; Julius et al. (2000) BioTechniques 28:702-708;Pizzato et al. (1998) Gene Therapy 5:1003-1007; Lopez de Quinto et al.(1998) Gene 217:51-56; WO 00/65034; WO 00/16800; U.S. Pat. No.5,854,037; U.S. Pat. No. 5,935,819; Bonaldo et al. (2000) Mem. Inst.Oswaldo Cruz 95:215-223.

SUMMARY OF THE INVENTION

[0009] The present invention provides live, replication-competent,recombinant bicistronic flaviviruses comprising, in order from 5′ to 3′,a 5′ untranslated region (UTR) from the parent flavivirus, an openreading frame (ORF) encoding all viral proteins of the parentflavivirus, an internal ribosome entry site (IRES), an exogenous nucleicacid molecule, and a 3′ UTR from the parent flavivirus. In general, therecombinant bicistronic flaviviruses of the invention are replicationcompetent and are attenuated, e.g., they are live, attenuated viruses.In some embodiments, the flavivirus is a yellow fever virus.Accordingly, in some embodiments, the invention provides recombinantyellow fever viruses (YFV), particularly live attenuated recombinantYFV, which comprise an exogenous (i.e., non-YFV) nucleotide sequencewhich encodes an exogenous (i.e., non-YFV) polypeptide. Infection ofhost cell with a recombinant bicistronic flavivirus provides forexpression of the exogenous nucleic acid in a host cell and productionof a polypeptide encoded by the exogenous nucleic acid. Such recombinantflavivirus are useful in eliciting an immune response to the exogenouspolypeptide.

[0010] The recombinant live attenuated flavivirus express an exogenousnucleotide sequence which encodes an exogenous polypeptide, such as, butnot limited to, a polypeptide obtained from a pathogenic agent otherthan the parent flavivirus used to generate the bicistronic flavivirus,a tumor antigen, or any other protein that has therapeutic activity.These recombinant flavivirus are useful, when introduced into amammalian subject, in eliciting an immune response to the exogenouspolypeptide in the subject. Thus, the recombinant flavivirus of theinvention serve as immunization vehicles.

[0011] The invention provides methods of delivering a polypeptide to ahost. The methods generally involve introducing a recombinantbicistronic flavivirus of the invention into a susceptible host. A widevariety of polypeptides can be expressed by the recombinant flavivirusof the invention, including those of microbial pathogens (e.g.,bacteria, protozoa, viruses (other than the parent flavivirus used togenerate the recombinant flavivirus of the invention), yeast, fungi, andthe like); proteins having therapeutic activity; and tumor-associatedantigens. In general, following infection of a host cell by therecombinant virus of the invention, the IRES site initiates translationof the exogenous polypeptide independently from translation of the viralpolyprotein precursor. The exogenous polypeptide may then be exported tothe host cell surface, may be presented on the cell surface as a peptidewith a major histocompatibility antigen, may be secreted from the cell,or may remain in the cytoplasm of the cell.

[0012] The invention provides pharmaceutical compositions comprisingrecombinant flavivirus of the invention. Such compositions can be used,for example, to reduce the severity of disease, reduce the risk ofclinical disease, prevent the onset of a disease and/or to amelioratethe disease via recruitment of the host immune system, and to deliver anexogenous polypeptide to a host.

[0013] The invention also provides methods of eliciting an immuneresponse to an antigen in a mammalian subject. Such methods compriseadministering a recombinant flavivirus of the invention to a mammaliansubject so as to elicit an immune response to the exogerious polypeptide(i.e., the antigen). The antigen can be a host antigen or an antigen ofa microorganism (e.g., bacteria, protozoa, viruses (other than theparent flavivirus used to generate the recombinant flavivirus of theinvention), yeast, fungi, and the like).

[0014] The invention also provides methods of delivering a polypeptidehaving therapeutic activity to a host. Such methods generally involveadministering a recombinant bicistronic flavivirus of the invention to amammalian host. The exogenous polypeptide is then produced in a hostcell. The therapeutic protein remains inside the cell, becomesassociated with a cell membrane, or is secreted from the cell.

[0015] In the context of tumor immunotherapy, expression of an exogenouspolypeptide in a host elicits an immune response to the tumor, with theresult that the tumor cell mass and/or tumor cell number is reduced,development of a tumor is prevented or delayed, and/or the probabilitythat a tumor will develop is reduced. Accordingly, the invention furtherprovides methods of reducing or inhibiting tumor cell growth, andmethods of reducing tumor cell mass and/or tumor cell numbers. Suchmethods comprise administering a recombinant flavivirus of the inventionwhich comprises exogenous sequences encoding a tumor-associated antigen(TAA)/epitope to a host bearing a tumor, such that the recombinantflavivirus enters a cell of the host and the exogenous TAA polypeptideis expressed on the surface of a host cell, is presented in the contextof an MHC molecule, or, alternatively, secreted from the host cell. Animmune response is elicited to a tumor which bears on its surface anantigen which comprises the exogenous TAA polypeptide or which resemblesthe exogenous polypeptide sufficiently to elicit an immune responsetoward the tumor cell. The immune response to the tumor bearing thetumor-associated antigen on its surface is sufficient to reduce,inhibit, or eliminate the tumor.

[0016] The invention further provides methods of preventing tumor cellgrowth, and methods of reducing the probability that a tumor will form,comprising administering a recombinant flavivirus of the invention,which comprises a tumor-associated antigen/epitope-encoding nucleicacid, to a host not bearing a tumor, such that the recombinantflavivirus enters a host cell, the tumor-associated antigen is expressedon the host cell surface and/or presented in the context of an MHCmolecule (e.g., MHC Class I), and an immune response is elicited to thetumor-associated antigen. The immune response to the tumor-associatedantigen is sufficient to prevent, or reduce the likelihood, of tumordevelopment in the host.

[0017] The invention further provides methods of producing an exogenouspolypeptide. The methods generally involve introducing a recombinantbicistronic flavivirus of the invention into a susceptible host cell(e.g., by infection), under conditions that favor production of theexogenous polypeptide by the host cell. In some embodiments, the methodsfurther comprise purifying the exogenous polypeptide.

[0018] A primary object of the invention is to provide a recombinantflavivirus that provides for production of an exogenous polypeptide in amammalian host. An object of the invention is to provide for productionof an exogenous polypeptide in a mammalian host that is suitable forinduction of an immune response to the polypeptide in a host followinginfection with the recombinant flavivirus. Such exogenous polypeptidesinclude, but are not limited to, an antigen produced by the host (e.g.,a tumor antigen), or an antigen from a microorganism (e.g., bacteria,protozoa, viruses (other than the parent flavivirus used to generate therecombinant flavivirus of the invention), yeast, fungi, and the like). Afurther object of the invention is to provide for production of anexogenous polypeptide in a host, where the exogenous polypeptideprovides a therapeutic activity.

[0019] An advantage of the invention is that the encoded exogenouspolypeptide is expressed independently of the viral polyproteinprecursor. Thus, the exogenous polypeptide undergoes intracellularco-translational and post-translational modifications in a manner thatis substantially identical to the processing it undergoes in its nativeenvironment. Therefore, the exogenous polypeptide exhibitscharacteristics of the native protein, e.g., the protein as it isproduced in its natural environment.

[0020] A further advantage of the invention is that, because theexogenous polypeptide is synthesized independently of the viralproteins, production of the exogenous polypeptide does not interferewith folding of the viral proteins, or with viral function.

[0021] An advantage of the invention is that the recombinant bicistronicflavivirus of the invention are live attenuated virus, which willcontinue to propagate until the intervention of the host's immunesystem.

[0022] Another advantage of the invention is that the recombinantbicistronic flavivirus exhibits low toxicity in vivo. Furthermore, thesubject recombinant bicistronic flaviviruses are safe, i.e., they arenon-pathogenic and thus do not produce a disease that wild-typeflaviviruses might produce.

[0023] Yet another advantage of the invention is that the subjectrecombinant bicistronic flaviviruses can be used to deliver any protein(consistent with the upper size limits discussed herein) to a host.

[0024] Yet another advantage of the invention is that the subjectrecombinant bicistronic flaviviruses can be used to deliver a proteinhaving therapeutic activity to a host.

[0025] Yet another advantage of the invention is that the recombinantflavivirus can express a polypeptide inside the host cell, and thus canprovide for induction of an immune response, particularly a cellularimmune response that involves, for example, antigen-specific cytotoxic Tlymphocytes (CTLs).

[0026] Yet another advantage of the invention is that the immuneresponse elicited using the recombinant flavivirus of the invention isnot limited to the infected cells, as the immune system will alsorecognize cells bearing the antigen expressed by the recombinantflavivirus. For example, the production of a tumor antigen by therecombinant flavivirus can “break immune tolerance” to tumor antigens,and induce an immune response against the tumor effective to, forexample, inhibit tumor growth.

[0027] These and other objects, advantages, and features of theinvention will become apparent to those persons skilled in the art uponreading the details of the invention as more fully described below.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0028]FIG. 1 is a schematic diagram of a recombinant bicistronic YFVvector for the expression of foreign proteins in accordance with theinvention.

[0029]FIG. 2 is a graphical representation of the production ofluciferase by bicistronic YFV vectors bYF-M-Luc, bYF-C-Luc, andbYF-G-Luc.

[0030]FIG. 3 is a graphical representation of the time course ofluciferase production in BHK cells transfected with bYF-M-Luc,bYF-C-Luc, or bYF-G-Luc

[0031]FIG. 4 shows the growth curve of bicistronic YF viruses expressingluciferase.

[0032] FIGS. 5A-D depict recombinant bicistronic YFV.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Before the present invention is described, it is to be understoodthat this invention is not limited to particular embodiments described,as such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

[0034] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

[0035] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

[0036] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a recombinant flavivirus” includes a plurality of such flaviviruses andreference to “the exogenous polypeptide” includes reference to one ormore exogenous polypeptides and equivalents thereof known to thoseskilled in the art, and so forth.

[0037] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

[0038] Further, the dates of publication provided may be different fromthe actual publication dates which may need to be independentlyconfirmed.

Definitions

[0039] The terms “polynucleotide” and “nucleic acid”, usedinterchangeably herein, refer to a polymeric forms of nucleotides of anylength, either ribonucleotides or deoxynucleotides.

[0040] Thus, this term includes, but is not limited to, single-,double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNAhybrids, or a polymer comprising purine and pyrimidine bases or othernatural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases.

[0041] The terms “peptide,” “oligopeptide,” “polypeptide,”“polyprotein,” and “protein” are used interchangeably herein, and referto a polymeric form of amino acids of any length, which can includecoded and non-coded amino acids, chemically or biochemically modified orderivatized amino acids, and polypeptides having modified peptidebackbones.

[0042] “Recombinant,” as used herein, means that a particular DNAsequence is the product of various combinations of cloning, restriction,and/or ligation steps resulting in a construct having a structuralcoding sequence distinguishable from homologous sequences found innatural systems. Generally, DNA sequences encoding the structural codingsequence can be assembled from cDNA fragments and short oligonucleotidelinkers, or from a series of oligonucleotides, to provide a syntheticgene which is capable of being expressed in a recombinanttranscriptional unit. Such sequences can be provided in the form of anopen reading frame uninterrupted by internal nontranslated sequences, orintrons, which are typically present in eukaryotic genes. Genomic DNAcomprising the relevant sequences could also be used. Sequences ofnon-translated DNA may be present 5′ or 3′ from the open reading frame,where such sequences do not interfere with manipulation or expression ofthe coding regions. Thus, e.g., the term “recombinant” polynucleotide ornucleic acid refers to one which is not naturally occurring, or is madeby the artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by eitherchemical synthesis means, or by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques. Suchis usually done to replace a codon with a redundant codon encoding thesame or a conservative amino acid, while typically introducing orremoving a sequence recognition site. Alternatively, it is performed tojoin together nucleic acid segments of desired functions to generate adesired combination of functions.

[0043] By “construct” is meant a recombinant nucleic acid, generallyrecombinant DNA, which has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences.

[0044] Similarly, a “recombinant polypeptide” or “recombinantpolyprotein” refers to a polypeptide or polyprotein which is notnaturally occurring, or is made by the artificial combination of twootherwise separated segments of amino acid sequences. This artificialcombination may be accomplished by standard techniques of recombinantDNA technology, such as described above, i.e., a recombinant polypeptideor recombinant polyprotein may be encoded by a recombinantpolynucleotide. Thus, a recombinant polypeptide or recombinantpolyprotein is an amino acid sequence encoded by all or a portion of arecombinant polynucleotide.

[0045] As used herein, the term “exogenous polypeptide” refers to apolypeptide that is not normally associated with a parent flavivirus innature.

[0046] The term “bicistronic” refers to the existence in the recombinantflaviviruses of the invention of two unrelated cistrons which areexpressed from a single viral transcriptional unit. The first cistron isan open reading frame encoding all viral proteins, while the secondcistron initiates translation from an IRES element located between thetwo cistrons, and includes an open reading frame encoding an exogenouspolypeptide.

[0047] The term “internal ribosomal entry site” or “IRES” refers to aviral, cellular, or synthetic (e.g., a recombinant) nucleotide sequencewhich allows for initiation of translation of an mRNA at a site internalto (e.g., at a site 3′ of) a first coding region within the same mRNA orat a site 3′ of the 5′ end of the mRNA, to provide for translation of anoperably linked coding region located downstream of (i.e., 3′ of) theinternal ribosomal entry site. This makes translation independent of the5′ cap structure, and independent of the 5′ end of the mRNA. An IRESsequence provides necessary cis-acting sequences required for initiationof translation of an operably linked coding region.

[0048] The term “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic peptide to induce aspecific humoral and/or cellular immune response in a mammal. As usedherein, “antigenic amino acid sequence,” “antigenic polypeptide,” or“antigenic peptide” means an amino acid sequence that, either alone orin association with an accessory molecule (e.g., a class I or class IImajor histocompatibility antigen molecule), can elicit an immuneresponse in a mammal.

[0049] The terms “antigen” and “epitope” are well understood in the artand refer to the portion of a macromolecule which is specificallyrecognized by a component of the immune system, e.g., an antibody or aT-cell antigen receptor. Epitopes are recognized by antibodies insolution, e.g., free from other molecules. Epitopes are recognized byT-cell antigen receptor when the epitope is associated with a class I orclass II major histocompatibility complex molecule. A “CTL epitope” isan epitope recognized by a cytotoxic T lymphocyte (usually a CD8⁺ cell)when the epitope is presented on a cell surface in association with anMHC Class I molecule.

[0050] An “allergen” refers to a substance that can induce an allergicor asthmatic response in a susceptible subject.

[0051] As used herein the term “isolated” is meant to describe acompound of interest (e.g., a recombinant virus, a polypeptide, etc.)that is in an environment different from that in which the compoundnaturally occurs. “Isolated” is meant to include compounds that arewithin samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

[0052] By “subject” or “host” or “individual” or “patient,” which termsare used interchangeably herein, is meant any vertebrate subject,particularly a mammalian subject, e.g., a human. Other subjects mayinclude ungulates (including sheep, pigs, cattle, and goats), equinehosts, rodents, lagomorphs, and so on. Of particular interest are thosesubjects susceptible to infection by a flavivirus, e.g., subjects whocan support flavivirus replication.

[0053] A “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids and tissue samples.

[0054] The terms “treatment,” “treating,” and the like are used hereinto generally refer to obtaining a desired pharmacologic or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, e.g., a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it, e.g., reducing the risk that an individual will develop thedisease, reducing the severity of a disease symptom; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease.

[0055] Recombinant Bicistronic Flaviviruses

[0056] The present invention provides recombinant bicistronicflaviviruses, particularly live attenuated recombinant bicistronicflaviviruses, which comprise, in order from to 3′, a 5′UTR from theparent flavivirus; an ORF encoding all proteins of the parentflavivirus; an internal ribosome entry site (IRES), an exogenous nucleicacid molecule (i.e., a nucleic acid molecule from other than the parentflavivirus) which comprises a nucleotide sequence that encodes anexogenous polypeptide (i.e., a polypeptide from other than the parentflavivirus); and a 3′ UTR from the parent flavivirus. Such recombinantflavivirus are useful for delivering the encoded exogenous polypeptideto a mammalian host. Such recombinant flavivirus are also useful ineliciting an immune response to the exogenous polypeptide. Forsimplicity, “exogenous” is used throughout as exemplary of suchsequences, but is not intended to be limiting.

[0057] The subject invention provides compositions and methods forgenerating and using recombinant flaviviruses to express exogenousnucleic acid molecules and to produce exogenous polypeptides encoded bythe exogenous nucleic acid molecules. FIG. 1 is a schematic diagram of arecombinant bicistronic YFV vector for the production of foreignproteins. In FIG. 1, the bar represents recombinant YFV genomic RNA; thestructural and non-structural viral genes are indicated withincorresponding boxes, the exogenous nucleic acid molecule (e.g.Luc=luciferase) at the 3′ end of the viral genome is indicated by astriped box. The sequences immediately 5′ of the luciferase-encodingregion correspond to an IRES element that allows for the expression ofthe luciferase coding region.

[0058] Yellow fever virus (YFV) is one non-limiting example of aflavivirus. The following description of YFV is generally applicable toother flaviviruses. Yellow fever virus is an enveloped,positive-stranded RNA virus and a member of the flaviviridae genus. Theviral genome is approximately 11 kb in length and encodes a singlepolypeptide precursor that encodes 10 viral proteins.

[0059] Bicistronic recombinant flaviviruses of the invention comprise anIRES element and a nucleic acid encoding an exogenous polypeptideinserted at the 3′ end of the ORF encoding all proteins of the parentflavivirus. The IRES element is inserted after the ORF encoding allproteins of the parent flavivirus. The IRES element initiates theexpression of the encoded exogenous polypeptide independently of theviral proteins which are translated by the normal flaviviruscap-dependent initiation.

[0060] The starting flavivirus, which is subsequently modified toinclude the exogenous sequences, is also referred to herein as the“parent” flavivirus. The parent flavivirus can be a native flavivirus(either pathogenic or, preferably, non-pathogenic), an attenuatedflavivirus, a vaccine flavivirus strain, or a recombinant flavivirus. Inmany embodiments, the parent flavivirus is a live, attenuatedflavivirus. Any of a variety of strains of flavivirus can be used ingenerating recombinant flavivirus as described herein.

[0061] Flavivirus which are suitable for use in generating recombinantflavivirus of the invention include, but are not limited to, yellowfever virus (YFV); Dengue virus, including Dengue types 1-4; JapaneseEncephalitis virus; Murray Valley Encephalitis virus; St. LouisEncephalitis virus; West Nile virus; tick-borne encephalitis virus;Hepatitis C virus; Kunjin virus; Central European encephalitis virus;Russian spring-summer encephalitis virus; Powassan virus; KyasanurForest disease virus; and Omsk hemorrhagic fever virus. In manyembodiments, the flavirus used as the starting flavivirus to generaterecombinant flavivirus is YFV.

[0062] The nucleotide sequences of a number of YFV strains are availablein public databases, including, e.g., GenBank. An exemplary strain is“YFV 17D.” The nucleotide sequence of the YFV genome, as well as theamino acid sequence of the encoded viral polyprotein are found underGenBank Accession No. X03700, and are also described in Rice et al.((1985) Science 229:726-733), both of which are incorporated herein byreference in their entirety for the nucleotide and protein sequencesdisclosed therein. Production of yellow fever virions (viral particles)is well known in the art.

[0063] In many embodiments, the flavivirus nucleotide sequences of therecombinant flavivirus are wild-type, i.e., they are sequences found innature. In other embodiments, the flavivirus nucleotide sequencescontain one or more mutations compared to a wild-type flavivirus. Instill other embodiments, the flavivirus portion of the recombinantflavivirus is derived from two or more different flaviviruses, i.e., theflavivirus used to construct a recombinant flavivirus is a chimericflavivirus.

[0064] In general, an exogenous nucleic acid molecule(s) in therecombinant flaviviruses comprises a nucleotide sequence encoding anexogenous polypeptide. The exogenous nucleic acid sequence arepositioned 3′ to a viral and/or cellular IRES element for independentexpression of the encoded exogenous polypeptide, while viral proteinsare translated from the cap structure at the 5′ end of the genome. Theinsertion of the IRES element and the exogenous nucleic acid outside ofthe flavivirus ORF, insures that there is no disruption of flavivirusprotein function, and/or proteolytic processing of the viralpolypeptide, and/or viral replication. Whether viral replication isadversely affected can be determined using well-established techniques,including, but not limited to, a plaque assay, and a one-step growthcurve assay.

[0065] Unlike other vectors which will produce only one cycle of antigenexpression and/or which will stop expression without the intervention ofthe host immune system, the replication-competent recombinant virus ofthe invention will continue to propagate until the immune system issufficiently activated to halt the infection. This produces a strongerimmune response against the exogenous antigenic peptide produced fromthe flavivirus as compared to the immune response that would be elicitedusing conventional expression vectors (e.g., a viral replicon).

[0066] The recombinant flavivirus also exhibits low toxicity to a hostupon infection. For example, YF-17D is a very safe and effective liveviral vaccine, prepared from infected chicken embryos under standardsdeveloped by the World Health Organization. After vaccination, immunityis elicited within 10 days in over 95% of vaccines and neutralizingantibodies directed against the virus can be detected for more than 35years. The vaccine safety record is outstanding: serious adversereactions to YF-17D vaccine are extremely uncommon, and reversion towild type is virtually non-existent.

[0067] Any of a variety of naturally-occurring and synthetic (e.g.recombinant) IRES sequences can be used in the recombinant bicistronicflaviviruses of the invention. Naturally occurring IRES sequences areknown in the art and include, but are not limited to, IRES sequencesderived from mengovirus, bovine viral diarrhea virus (BVDV), hepatitis Cvirus (HCV; e.g., nucleotides 1202-1812 of the nucleotide sequenceprovided under GenBank Accession number AJ242654), GTX, Cyr61 a, Cyr61b,poliovirus, the immunoglobulin heavy-chain-binding protein (BiP),immunoglobulin heavy chain, a picomavirus, murine encephalomyocarditisvirus, poliovirus, and foot and mouth disease virus (e.g., nucleotidenumbers 600-1058 of the nucleotide sequence provided under GenBankAccession No. AF308157). Other IRES sequences such as those reported inWO 96/01324; WO 98/49334; WO 00/44896; and U.S. Pat. No. 6,171,821 canbe used in the recombinant flaviviruses of the invention.

[0068] Mutants, variants and derivatives of naturally occurring IRESsequences may be employed in the present invention provided they retainthe ability to initiate translation of an operably linked codingsequence located 3′ of the IRES. An IRES sequence suitable for use inthe present invention has at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, or more, nucleotide sequence identity with a naturally occurringIRES. An IRES sequence suitable for use in the present invention mayalso be a fragment of a naturally occurring IRES, provided the fragmentfunctions to allow ribosome attachment and initiate translation of anoperably linked 3′ coding region.

[0069] Additional features can be incorporated into the design of areplication-competent recombinant flavivirus virus of the invention,such as polylinker sequences (e.g., EcoR1, NotI, BssH2, and XhoI) tofacilitate the ease of insertion of desired foreign sequences into therecombinant vector.

[0070] More than one nucleic acid sequence encoding an exogenous proteinor polypeptide to be produced can be included in the recombinantreplication-competent flavivirus virus which, as a result, produces thecorresponding number of exogenous proteins or polypeptides. The two ormore nucleic acid sequences can each encode a different product or canencode the same product (e.g., if enhanced production of a protein orpolypeptide is desired).

[0071] The addition of an IRES element and an exogenous nucleic acid 3′of the viral genome can be accomplished by standard techniques ofmolecular biology, such as described in numerous standard protocoltexts, including e.g., Current Protocols in Molecular Biology, (F. M.Ausubel, et al., Eds. 1987, and updates. Example 1 provides furtherguidance for how particular recombinant flaviviruses were generated.Using these guidelines, any of a variety of IRES elements and exogenousnucleic acids can be added to the flavivirus genome.

[0072] The exogenous nucleic acid molecule is from about 12 to about 18,from about 15 to about 24, from about 21 to about 30, from about 30 toabout 60, from about 60 to about 90, from about 90 to about 120, fromabout 120 to about 150, from about 150 to about 180, from about 180 toabout 240, from about 240 to about 300, from about 300 to about 600,from about 600 to about 1200, from about 1200 to about 1500, from about1500 to about 2100, from about 2100 to about 2400, or from about 2400 toabout 3000 nucleotides in length.

[0073] The encoded exogenous polypeptide is from about 4 to about 6,from about 5 to about 8, from about 7 to about 10, from about 10 toabout 20, from about 20 to about 30, from about 30 to about 40, fromabout 40 to about 50, from about 50 to about 60, from about 60 to about80, from about 80 to about 100, from about 100 to about 200, from about200 to about 400, from about 400 to about 500, from about 500 to about700, from about 700 to about 800, or from about 800 to about 1000 aminoacids in length.

[0074] Recombinant bicistronic flaviviruses of the invention enter ahost cell, and subsequently the host cell produces the exogenouspolypeptide encoded by the recombinant flavivirus. In some embodiments,the exogenous polypeptide remains intracellular. In other embodiments,the exogenous polypeptide is associated with the cell surface. In stillother embodiments, the exogenous polypeptide is secreted from the cell.

[0075] Recombinant bicistronic flaviruses of the invention are usefulfor delivering a polypeptide to a host. A subject recombinantbicistronic flavivirus is administered to a mammalian host.Subsequently, the subject recombinant bicistronic flavivirus enters ahost cell, and the exogenous polypeptide is produced by the cell. Theexogenous polypeptide remains intracellular, becomes associated with acell membrane (e.g., the plasma membrane), or is secreted from the cell.

[0076] Exogenous polypeptides can be a polypeptide from any of a varietyof pathogenic organisms, including, but not limited to, viruses,bacteria, yeast, fungi, and protozoa; a tumor-associated antigen; a“self” antigens; a foreign antigen from other than a pathogenicmicroorganism; a protein that has a therapeutic activity; and the like.

[0077] Where the polypeptide comprises one or more antigenic epitopes,any nucleic acid molecule comprising a nucleotide sequence which encodesa polypeptide which, when produced by a cell infected by a recombinantflavivirus of the invention, increases an immune response is suitablefor use in the present invention. Nucleic acid sequences encoding one ormore exogenous polypeptides (e.g., antigens or epitopes) of interest canbe included in a recombinant bicistronic flavivirus of the invention. Ifmore than one exogenous antigen or epitope of interest is encoded by theexogenous nucleic acid sequences, they can be antigens or epitopes of asingle pathogen or antigens or epitopes from more than one (different)pathogen. In many embodiments, such an organism is a pathogenicmicroorganism. For example, such an exogenous epitope may be found onbacteria, parasites, viruses, yeast, or fungi that are the causativeagents of diseases or disorders. In other embodiments, the antigen is anallergen. In still other embodiments, the antigen is a tumor-associatedantigen. In still other embodiments, the antigen is a sperm-associatedantigen. In further embodiments, the antigen is one that producesadverse physiological effects in a vertebrate host, including, but notlimited to, a venom.

[0078] Pathogenic viruses include, but are not limited to, Retroviridae(e.g., human immunodeficiency viruses, such as HIV-1 (also referred toas HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and other isolates, suchas HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g.,dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Bimaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses); Poxyiridae (variola viruses, vaccinia viruses, pox viruses);and Iridoviridae (e.g., African swine fever virus); Hepatitis C virus;and unclassified viruses (e.g., the agent of delta hepatitis (thought tobe a defective satellite of hepatitis B virus); Norwalk and relatedviruses, and astroviruses).

[0079] Pathogenic bacteria include, but are not limited to,Helicobacterpyloris, Borelia burgdorferi, Legionella pneumophila,Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyrogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus anthracis, Corynebacterium diphtheriae,Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, pathogenic strains of Escherichia coli, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli.

[0080] Infectious fungi include, but are not limited to, Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, Candida albicans.

[0081] Infectious protozoa include, but are not limited to, Plasmodiumspp., e.g., Plasmodium falciparum; Trypanosomes, e.g., Trypanosomacruzi; and Toxoplasma gondii.

[0082] Allergens include, but are not limited to, pollens, insectvenoms, animal dander dust, fungal spores and drugs (e.g. penicillin).Examples of natural, animal and plant allergens include proteinsspecific to the following genera: Canine (Canis familiaris);Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felisdomesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Loliumperenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica);Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinosa);Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa);Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata);Parietaria (e.g. Parietaria officinalis or Parietaria judaica);Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum);Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica andCupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperusvirginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuyaorientalis); Chamaecyparis (e.g. Charnaecyparis obtusa); Periplaneta(e.g. Periplaneta americana); Agropyron(e.g. Agropyron repens); Secale(e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g.Dactylis glomerata); Festuca(e.g. Festuca elatior); Poa (e.g. Poapratensis or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g.Holcus lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum);Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis(e.g. Agrostisalba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis).

[0083] Any of a variety of known tumor-associated antigens (TAA) can beinserted into flavivirus of the invention. The entire TAA may be, butneed not be, inserted. Instead, a portion of a TAA, e.g., an epitope,particularly an epitope that is recognized by a CTL, may be inserted.Tumor-associated antigens (or epitope-containing fragments thereof)which may be inserted into flavivirus include, but are not limited to,MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, high molecular weightmelanoma-associated antigen MAA, GD2, carcinoembryonic antigen (CEA),TAG-72, ovarian-associated antigens OV-TL3 and MOV 18, TUAN, alpha-fetoprotein (AFP), OFP, CA-125, CA-50, CA-19-9, renal tumor-associatedantigen G250, EGP-40 (also known as EpCAM), S100 (malignantmelanoma-associated antigen), p53, prostate tumor-associated antigens(e.g., PSA and PSMA), and p21ras.

[0084] Other antigens of interest include, but are not limited to,sperm-associated antigens, venoms, hormones, and the like.Sperm-associated proteins are known in the art, and a nucleic acidmolecule encoding any such protein is suitable for use herein. See,e.g., Primakoff (1994) Reproductive Immunol. 31:208-210; Naz et al.(1995) Human Reprod. Update 1:1-18; Kerretal. (1998) J. Reprod. Immunol.40:103-118; and U.S. Pat. No. 6,197,940. Hormones of interest include,but are not limited to, human chorionic gonadotrophin (hCG). Hormonessuch as hCG are useful to elicit specific antibodies, for use ascontraceptive. Venoms of interest include those from any poisonousanimal, e.g., snake venoms, including, but not limited to,α-neurotoxins, kappa toxins, β-neurotoxins, dendrotoxins, cardiotoxins,myotoxins, and hemorrhaging. Of particular interest in many embodimentsare modified venoms that elicit specific antibodies, but are notthemselves toxic. Such modified venoms are useful to elicit an immuneresponse to a venom, and in many embodiments, elicit a protective immuneresponse such that, upon subsequent exposure to the venom from an animalsource, any adverse physiological effects of the venom are mitigated.

[0085] A “therapeutic protein” includes a protein that the host does notproduce but is in need of; a protein that the host does not normallyproduce, but which has a therapeutic activity; a protein that the hostproduces, but produces in inadequate amounts; a protein that the hostproduces but in a form which is inactive, or which has reduced activitycompared with an activity normally associated with the protein; or aprotein that the host produces in adequate amounts and with normalactivity associated with that protein. Therapeutic proteins includenaturally-occurring proteins, and recombinant proteins whose amino acidsequences differ from a naturally-occurring counterpart protein, whichrecombinant proteins have substantially the same, an altered activity,or enhanced activity relative to a naturally-occurring protein. Proteinsthat have therapeutic activity include, but are not limited to,cytokines, including, but not limited to, interleukins, endothelin,colony stimulating factors, tumor necrosis factor, and interferons;hormones, including, but not limited to, a growth hormone, insulin;growth factors, including, but not limited to human growth factor,insulin-like growth factor; bioactive peptides; trophins; neurotrophins;soluble forms of a membrane protein including, but not limited to,soluble CD4; enzymes; regulatory proteins; structural proteins; clottingfactors, including, but not limited to, factor XIII; chemokines;erythropoietin; tissue plasminogen activator; etc.

[0086] The nucleic acid molecule encoding the exogenous protein to beproduced by a host cell following infection of the host cell by therecombinant flavivirus to the present invention can be obtained bytechniques known in the art, including but not limited to, chemical orenzymatic synthesis, purification from genomic DNA of the microorganism,by purification or isolation from a cDNA encoding the exogenous protein,by cDNA synthesis from RNA of an organism, or by standard recombinantmethods (Maniatis et al., Molecular Cloning, A Laboratory Manual, 1982,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Nucleotidesequences encoding many of the above-listed exogenous proteins arepublicly available. Variant of such sequences can readily be generatedby those skilled in the art using standard recombinant methods,including site-directed and random mutagenesis. The nucleic acidmolecule encoding the exogenous polypeptide can further includesequences that direct secretion of the protein from the cell, sequencesthat alter RNA and/or protein stability, and the like.

[0087] Compositions Comprising Recombinant Bicistronic Flaviviruses ofthe Invention

[0088] The present invention further provides compositions, includingpharmaceutical compositions, comprising a recombinant flavivirus of theinvention.

[0089] Compositions comprising a recombinant flavivirus of the inventionmay include a buffer, which is selected according to the desired use ofthe recombinant flavivirus, and may also include other substancesappropriate to the intended use. Where the intended use is to elicit orincrease an immune response, the composition is referred to as “animmunogenic composition.” Those skilled in the art can readily select anappropriate buffer, a wide variety of which are known in the art,suitable for an intended use. In some instances, the composition cancomprise a pharmaceutically acceptable excipient, a variety of which areknown in the art and need not be discussed in detail herein.Pharmaceutically acceptable excipients have been amply described in avariety of publications, including, for example, A. Gennaro (2000)“Remington: The Science and Practice of Pharmacy”, 20th edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds 7^(th) ed., Lippincott,Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^(rd) ed. Amer. Pharmaceutical Assoc.

[0090] Pharmaceutical Formulations, Routes of Administration, andDosages

[0091] Formulations

[0092] The invention further provides pharmaceutical formulationscomprising a recombinant bicistronic flavivirus of the invention.Pharmaceutical formulations can comprise more than one differentrecombinant flavivirus, e.g., recombinant flaviviruses comprisingdifferent exogenous nucleic acid molecules.

[0093] Pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, injectableformulations, suspensions, sprays, suppositories, transdermalapplications (e.g., patches, etc.), salves, lotions and the like.Pharmaceutical grade organic or inorganic carriers and/or diluentssuitable for oral and topical use can be used to make up compositionscontaining the therapeutically active compounds. Diluents known to theart include aqueous media, vegetable and animal oils and fats.Stabilizing agents, wetting and emulsifying agents, salts for varyingthe osmotic pressure or buffers for securing an adequate pH value, andskin penetration enhancers can be used as auxiliary agents.

[0094] Pharmaceutical grade organic or inorganic carriers and/ordiluents suitable for oral and topical use can be used to make upcompositions containing the therapeutically-active compounds. Diluentsknown to the art include aqueous media, vegetable and animal oils andfats. Stabilizing agents, wetting and emulsifying agents, salts forvarying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents ororal preparations, the formulations can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0095] The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

[0096] The agents can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

[0097] Furthermore, the agents can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The compounds of the present invention can be administeredrectally via a suppository. The suppository can include vehicles such ascocoa butter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

[0098] When used as an immunogenic composition (e.g., a “vaccine”), arecombinant flavivirus of the invention can be formulated in a varietyof ways. In general, the immunogenic composition of the invention isformulated according to methods well known in the art of vaccinepreparation, using suitable pharmaceutical carrier(s) and/or vehicle(s).A suitable vehicle is sterile saline. Other aqueous and non-aqueousisotonic sterile injection solutions and aqueous and non-aqueous sterilesuspensions known to be pharmaceutically acceptable carriers and wellknown to those of skill in the art may be employed for this purpose.

[0099] Optionally, a vaccine composition (“immunogenic composition”) ofthe invention may be formulated to contain other components, including,e.g., adjuvants, stabilizers, pH adjusters, preservatives and the like.Such components are well known to those of skill in the vaccine art.Adjuvants include, but are not limited to, aluminum salt adjuvants(Nicklas (1992) Res. Immunol. 143:489-493); saponin adjuvants; Ribi'sadjuvants (Ribi ImmunoChem Research Inc., Hamilton, Mont.); MontanideISA adjuvants (Seppic, Paris, France); Hunter's TiterMax adjuvants(CytRx Corp., Norcross, Ga.); Gerbu adjuvants (Gerbu Biotechnik GmbH,Gaiberg, Germany); and nitrocellulose (Nilsson and Larsson (1992) Res.Immunol. 143:553-557). In addition, other components that may modulatean immune response may be included in the formulation, including, butnot limited to, cytokines, such as interleukins; colony-stimulatingfactors (e.g., GM-CSF, CSF, and the like); and tumor necrosis factor.

[0100] Routes of Administration

[0101] Conventional and pharmaceutically acceptable routes ofadministration include intranasal, intramuscular, intratracheal,intratumoral, subcutaneous, intradermal, vaginal, intrapulmonary,intravenous, rectal, nasal, oral and other parenteral routes ofadministration. Routes of administration may be combined, if desired, oradjusted depending upon the antigenic peptide or the disease. Thecomposition can be administered in a single dose or in multiple doses,and may encompass administration of booster doses, to elicit and/ormaintain immunity.

[0102] When they are used as vaccines, the recombinant flavivirus of thepresent invention are administered to an individual using known methods.They will generally be administered by the same routes by whichconventional (presently-available) vaccines are administered and/or byroutes that mimic the route by which infection by the pathogen ofinterest occurs.

[0103] Dosages

[0104] The recombinant flavivirus vaccine is administered in an“effective amount” that is, an amount effective to achieve production ofthe exogenous polypeptide in the host at a desired level. In general, itis expected that each dose of recombinant bicistronic flavivirus will besufficient to generate, upon infection of host cells, about 1-1000 μg ofprotein, generally from about 1-200 μg, normally from about 10-100 μg.The dose of recombinant bicistronic flavivirus administered to anindividual will generally be in a range of from about 10² to about 10⁷,from about 10³ to about 10⁶, or from about 10⁴ to about 10⁵ plaqueforming units (PFU).

[0105] In some embodiments, an “effective amount” of a subjectrecombinant bicistronic flavivirus is an amount sufficient to achieve adesired therapeutic effect.

[0106] In some embodiments, an “effective amount” of a subjectrecombinant bicistronic flavivirus is an amount of recombinantflavivirus that is effective in a selected route of administration toelicit an immune response to the exogenous antigen.

[0107] In many embodiments, e.g., where the exogenous polypeptide is oneassociated with a pathogenic microorganism, an “effective amount” is anamount that is effective to facilitate protection of the host againstinfection, or symptoms associated with infection, by a pathogenicorganism, e.g., to reduce a symptom associated with infection, and/or toreduce the number of infectious agents in the individual. In theseembodiments, an effective amount reduces a symptom associated withinfection and/or reduces the number of infectious agents in anindividual by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or at least about 90%, or more,when compared to the symptom or number of infectious agents in anindividual not treated with the recombinant bicistronic flavivirus, ortreated with the parent flavivirus. Symptoms of infection by apathogenic microorganism, as well as methods for measuring suchsymptoms, are known in the art. Methods for measuring the number ofpathogenic microorganisms in an individual are standard in the art.

[0108] In some embodiments, e.g., where the exogenous polypeptide is atumor-associated antigen, an “effective amount” of a recombinantflavivirus vaccine is an amount of recombinant flavivirus that iseffective in a route of administration to elicit an immune responseeffective to reduce or inhibit tumor cell growth, to reduce tumor cellmass or tumor cell numbers, or to reduce the likelihood that a tumorwill form. In these embodiments, an effective amount reduces tumorgrowth and/or the number of tumor cells in an individual by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or at least about 90%, or more, when compared to the tumorgrowth and/or number of tumor cells in an individual not treated withthe recombinant bicistronic flavivirus, or treated with the parentflavivirus. Methods of measuring tumor growth and numbers of tumor cellsare known in the art.

[0109] In other embodiments, e.g., where the exogenous polypeptide is asperm-associated antigen, or a hormone such as hCG, an “effectiveamount” of a recombinant bicistronic flavivirus is an amount effectiveto reduce fertility in the individual. In these embodiments, aneffective amount reduces fertility in an individual by at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90%, or more, when compared to fertility in anindividual not treated with the recombinant bicistronic flavivirus, ortreated with the parent flavivirus.

[0110] In still other embodiments, e.g., where the exogenous polypeptideis a venom or a modified venom, an “effective amount” of a recombinantbicistronic flavivirus is an amount effective to reduce the magnitude ofan adverse physiological effect of subsequent exposure to the venom froman animal source. In these embodiments, an effective amount reduces anadverse physiological effect associated with exposure to an animal venomin an individual by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, or at least about 90%, or more,when compared to the physiological effect of the animal venom in anindividual not treated with the recombinant bicistronic flavivirus, ortreated with the parent flavivirus.

[0111] The amount of recombinant flavivirus in each dose is selected asan amount which induces an immune response to the encoded exogenousantigen, and/or which induces an immunoprotective or otherimmunotherapeutic response without significant, adverse side effectsgenerally associated with typical vaccines. Such amount will varydepending upon which specific exogenous polypeptide is employed, whetheror not the vaccine formulation comprises an adjuvant, and a variety ofhost-dependent factors.

[0112] An effective dose of recombinant flavivirus nucleic acid-basedvaccine will generally involve administration of from about 1-1000 μg ofnucleic acid. Alternatively, an effective dose of recombinant flavivirusvaccine will generally be in a range of from about 10² to about 10⁷,from about 10³ to about 10⁶, or from about 10⁴ to about 10⁵ plaqueforming units (PFU). An optimal amount for a particular vaccine can beascertained by standard studies involving observation of antibody titersand other responses in subjects. The levels of immunity provided by thevaccine can be monitored to determine the need, if any, for boosters.Following an assessment of antibody titers in the serum, optionalbooster immunizations may be desired. The immune response to the proteinof this invention is enhanced by the use of adjuvant and/or animmunostimulant.

[0113] Uses of Recombinant Flaviviruses of the Invention

[0114] Recombinant flaviviruses of the invention are useful to deliver apolypeptide to a mammalian host; and to elicit or increase an immuneresponse to an antigen encoded by the recombinant flavivirus.Recombinant flavivirus of the present are also useful for producing theexogenous polypeptide in host cells, such as mammalian, particularlyhuman, cells or other cell types. The exogenous protein can further beisolated or purified using standard methods.

[0115] Methods of Using the Recombinant Flaviviruses of the Invention

[0116] The present invention provides methods for delivering atherapeutic polypeptide to a mammalian host; methods for eliciting animmune response to an antigen in a vertebrate host; and methods forproducing an exogenous polypeptide in a vertebrate host cell

[0117] Methods for Delivering a Therapeutic Polypeptide to a MammalianHost

[0118] The present invention provides methods for delivering apolypeptide to a mammalian host. The methods generally involveadministering a recombinant bicistronic flavivirus of the invention to avertebrate host, wherein the flavivirus enters a host cell, theexogenous polypeptide is expressed independently of the viral proteins.In some embodiments, the exogenous polypeptide remains intracellular. Inother embodiments, the exogenous polypeptide becomes associated with theplasma membrane of a host cell. In other embodiments, the exogenouspolypeptide is secreted from the cell. In those embodiments in which theexogenous polypeptide is secreted from the cell, the exogenouspolypeptide can be secreted into the extracellular milieu, e.g., theinterstitial fluid; and/or the exogenous polypeptide can enter the bloodstream; and/or the exogenous polypeptide can bind to and/or enter a cellother than the cell in which it was produced.

[0119] In many embodiments, the exogenous polypeptide is one that hastherapeutic activity, such that when the protein is produced in themammalian host, a therapeutic effect is achieved. Whether, and at whatlevel, a therapeutic protein is produced in an individual is readilydetermined using any known method, e.g., methods for detecting thepresence of and/or measuring the amount of a protein, including, but notlimited to, an enzyme-linked immunosorbent assay, a radioimmunoassay,and the like, using specific antibody; and methods for detecting thepresence of and/or measuring the amount of a biological activityassociated with the protein. Whether a therapeutic effect is achievedcan be determined using a method appropriate to the particulartherapeutic effect. For example, whether a therapeutic effect isachieved when insulin is delivered to a host using the subject methodcan be determined by measuring glucose levels in the individual.

[0120] Methods of delivering a therapeutic protein to a mammalian hostare appropriate for treating a mammalian subject having an inherited oracquired disease associated with a specific protein deficiency (e.g.,diabetes, hemophilia, anemia, severe combined immunodeficiency). Suchprotein deficient states are amenable to treatment by replacementtherapy, i.e., expression of a protein to restore the bloodstream levelsof the protein to at least normal levels.

[0121] Alternatively, the mammalian subject may have a condition whichis amenable to treatment by expression or over-expression of a proteinwhich is either normally present in a healthy mammalian subject or isforeign to the mammalian subject. The subject methods are also useful toenhance expression of a protein present in a normal mammal, or toexpress a protein not normally present in a normal mammal, in order toachieve a desired effect (e.g., to enhance a normal metabolic process).The methods are also useful to provide for production of a protein in acell that is defective in the host.

[0122] Methods of Increasing an Immune Response to an ExogenousPolypeptide

[0123] The present invention provides methods for eliciting an immuneresponse to an antigen. The methods generally involve administering arecombinant bicistronic flavivirus of the invention to a vertebratehost, wherein the flavivirus enters a host cell, the exogenouspolypeptide is expressed independently of the viral proteins, and animmune response is elicited to the exogenous polypeptide.

[0124] In some embodiments, recombinant flavivirus as described hereinare useful for inducing or increasing an immune response to an antigenin an individual. When the exogenous polypeptide is produced in avertebrate host, it elicits an immune response to the exogenouspolypeptide. In many embodiments, the immune response protects against acondition or disorder caused by or associated with expression of or thepresence in the host of, an antigen comprising the epitope. In someembodiments the antigen is a pathogen-associated antigen, and the immuneresponse provides protection against challenge or infection by theexogenous pathogen (bacterial, viral, fungal, parasitic) in which theantigen occurs. Recombinant bicistronic flaviviruses of the inventionare, therefore, useful as vaccines (also referred to herein as“immunogenic compositions”) to elicit and/or increase an immune responseto the antigen. In other embodiments, the antigen is a tumor-associatedantigen, and the immune response to the antigen reduces the growth of acancer cell bearing the antigen, and/or reduces the number of tumorcells bearing the antigen. In other embodiments, the antigen is anallergen, and the immune response to the antigen reduces an allergicresponse to the antigen. In still other embodiments, the antigen is asperm-associated antigen, and the immune response to the antigen resultsin decreased fertility. In still other embodiments, the antigen is onethat produces adverse physiological effects in a vertebrate host, andthe immune response to the antigen mitigates the adverse physiologicaleffects.

[0125] In many embodiments, the exogenous polypeptide is an antigenicpolypeptide of a microbial pathogen. Such recombinant flavivirus canthen be administered to a host to prevent or treat infection by thepathogen, or to prevent or treat symptoms of such pathogenic infection.Of particular interest in some embodiments is the prevention ortreatment of infection or disease caused by microbial pathogens that,during the course of infection, are present intracellularly, e.g.,viruses (e.g., HIV), bacteria (e.g., Shigella, Listeria, mycobacteria,and the like), parasites (e.g., malarial parasites (e.g., Plasmodiumfalciparum), trypanosomes, and the like), etc. Antigenic polypeptides ofsuch microbial pathogens are well known in the art, and can be readilyselected for use in the present recombinant flavivirus vaccine by theordinarily skilled artisan.

[0126] In addition, a recombinant flavivirus of the invention can beused as a delivery vehicle to delivery any antigen to an individual, toprovoke an immune response to the antigen. In some embodiments,recombinant flavivirus of the invention are used as bivalent ormultivalent vaccine to treat human or veterinary diseases caused byinfectious pathogens, particularly viruses, bacteria, and parasites.Examples of epitopes which could be delivered to a host in a multivalentflavivirus vaccine of the invention include multiple epitopes fromvarious serotypes of Group B streptococcus, influenza virus, rotavirus,and other pathogenic organisms known to exist in nature in multipleforms or serotypes; epitopes from two or more different pathogenicorganisms; and the like.

[0127] Suitable subjects include naïve subjects (i.e., subjects who werenever exposed to the antigen such that the antigen or pathogen enteredthe body), and subjects who were previously exposed to the antigen, butdid not mount a sufficient immune response to the pathogenic organism.

[0128] Whether an immune response has been elicited to a pathogenicorganism can be determined (quantitatively, e.g., by measuring aparameter, or qualitatively, e.g., by assessing the severity of asymptom, or by detecting the presence of a particular parameter) usingknown methods. Methods of measuring an immune response are well known inthe art and include enzyme-linked immunosorbent assay (ELISA) fordetecting and/or measuring antibody specific to a given pathogenicorganism; and in vitro assays to measure a cellular immune response(e.g., a CTL assay using labeled, inactivated cells expressing theepitope on their cell surface with MHC Class I molecules). Whether animmune response is effective to facilitate protection of the hostagainst infection, or symptoms associated with infection, by apathogenic organism can be readily determined by those skilled in theart using standard assays, e.g., determining the number of pathogenicorganisms in a host (e.g., measuring viral load, and the like);measuring a symptom caused by the presence of the pathogenic organism inthe host (e.g., body temperature, CD4⁺ T cell counts, and the like).

[0129] In other embodiments, a polypeptide antigen expressed on a giventumor cell (e.g., a tumor associated antigen; “TAA”) is inserted into arecombinant flavivirus of the invention as described herein. Suchrecombinant flavivirus can be administered to an individual having, orsuspected of having, a tumor. In some cases, such recombinant flaviviruscan be administered to an individual who does not have a tumor, but inwhom protective immunity is desired. As is often the case, the immunesystem does not mount an immune response effective to inhibit orsuppress tumor growth, or eliminate a tumor altogether. Tumor-associatedantigens are often poorly immunogenic; perhaps due to an active andongoing immunosuppression against them. Furthermore, cancer patientstend to be immunosuppressed, and only respond to certain T-dependentantigens. In these cases, introduction into the host of a recombinantflavivirus of the invention which expresses an exogenous peptidecorresponding to an antigen expressed on the tumor cell surface canelicit an immune response to the tumor in the host.

[0130] Any of a variety of known tumor-associated antigens (TAA) can beinserted into flavivirus of the invention. The entire TAA may be, butneed not be, inserted. Instead, a portion of a TAA, e.g., an epitope,particularly an epitope that is recognized by a CTL, may be inserted.Tumor-associated antigens (or epitope-containing fragments thereof)which may be inserted into flavivirus include, but are not limited to,MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, high molecular weightmelanoma-associated antigen MAA, GD2, carcinoembryonic antigen (CEA),TAG-72, ovarian-associated antigens OV-TL3 and MOV18, TUAN, alpha-fetoprotein (AFP), OFP, CA-125, CA-50, CA-19-9, renal tumor-associatedantigen G250, EGP-40 (also known as EpCAM), S100 (malignantmelanoma-associated antigen), p53, prostate tumor-associated antigens(e.g., PSA and PSMA), and p21ras.

[0131] Suitable subjects include subjects who do not have cancer, butare considered at risk of developing cancer; and subjects who havecancer, but who have not mounted an immune response sufficient to reduceor eliminate the cancer.

[0132] Recombinant flavivirus comprising a TAA can be administered to anindividual as described above. Whether an immune response is elicited toa given tumor can be determined by methods standard in the art,including, but not limited to, assaying for the presence and/or amountof TAA-specific antibody in a biological sample derived from theindividual, e.g., by enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and the like; assaying for the presence and/ornumbers of CTLs specific for a TAA; and the like. Assays for determiningthe presence and/or numbers of TAA-specific CTLs are known in the artand include, but are not limited to, chromium-release assays, tritiatedthymidine incorporation assays, and the like. Standard immunologicalprotocols may be used, which can be found in a variety of texts,including, e.g., Current Protocols in Immunology (J. E. Coligan, A. M.Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober Eds. 1991).

[0133] Whether an immune response is effective in reducing the number oftumor cells in an individual can be determined by standard assays,including, but not limited to, measuring tumor cell mass, measuringnumbers of tumor cells in an individual, and measuring tumor cellmetastasis. Such assays are well known in the art and need not bedescribed in detail herein.

[0134] Using the methods and compositions described herein in connectionwith the subject invention, an immunoprotective response againstmicrobial infection can be induced in any subject, human or non-human,susceptible to infection by a microbial pathogen, or who has beeninfected with a microbial pathogen. Where the recombinant flaviviruscomprises an exogenous nucleic acid sequence encoding a TAA, the subjectmay be one that is known to have cancer, is suspected of having cancer,or does not have cancer, but in whom immunity to cancer is to beinduced.

[0135] Methods for Producing an Exogenous Polypeptide

[0136] The invention further provides methods of producing an exogenouspolypeptide in a vertebrate host cell. The methods generally involvecontacting a recombinant bicistronic flavivirus of the invention with asusceptible host cell, culturing the host cell for a period of time toallow production of the exogenous polypeptide by the host cell. In someembodiments, the methods further comprise purifying the exogenouspolypeptide from the host cell or from the culture medium.

[0137] In some embodiments, the exogenous protein remains intracellular(e.g., in the cytoplasm, in a cell membrane, or in an organelle), inwhich case the cells are disrupted. A variety of protocols fordisrupting cells to release an intracellular protein are known in theart, and can be used to extract an exogenous protein from a cell. Inother embodiments, the exogenous protein is secreted into the medium inwhich the cells are grown.

[0138] In certain embodiments, an exogenous polypeptide is present in acomposition that is enriched for the exogenous polypeptide as comparedto its naturally occurring environment, or as compared to a startingmaterial comprising the exogenous polypeptide. For example, purifiedexogenous polypeptides are provided, where by purified is meant that theexogenous polypeptide is present in a composition that is substantiallyfree of proteins other than the exogenous polypeptide, where bysubstantially free is meant that less than 90%, usually less than 60%and more usually less than 50% of the composition is made up of proteinsother than the exogenous polypeptide. An exogenous polypeptide may alsobe present as an isolate, by which is meant that the protein issubstantially free of other proteins and other naturally occurringbiologic molecules, such as oligosaccharides, polysaccharides, lipids,polynucleotides and fragments thereof, and the like, where substantiallyfree in this instance means that less than 70%, usually less than 60%and more usually less than 50% of the composition containing theisolated protein is some other naturally occurring biological molecule.In certain embodiments, the exogenous polypeptide is present insubstantially pure form, where by substantially pure form is meant atleast 95%, usually at least 97% and more usually at least 99% pure.

[0139] Any convenient protein purification procedures may be employed,where suitable protein purification methodologies are described in Guideto Protein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may prepared from the infected host cell, or a cellculture supernatant may be produced, and the exogenous protein purifiedusing HPLC, exclusion chromatography, gel electrophoresis, affinitychromatography, and the like.

EXAMPLES

[0140] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations are used,e.g., h, hours; min, minutes; s, seconds; and the like.

[0141] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

Example 1 Engineering Bicistronic recombinant Yellow Fever Virus

[0142] Bicistronic YFV genomes were engineered by inserting at the 3′non-coding region of the YVF genome a fragment of 120 to 700 bpcorresponding to the internal ribosomal entry site (IRES) derived fromeither viral or cellular RNAs. Table 1 provides a list of IRES whichwere used to generate recombinant YFV. Translation of normal YFVproteins initiates from the 5′ capped end of the viral genome, whiletranslation of inserted foreign proteins is directed from the insertedIRES. All of the YFV proteins are correctly produced and viralreplication proceeds normally in the recombinant YFV of the invention.The replication characteristics of the recombinant viruses were assessedas well as the levels of antigen expression and genetic stability. TABLE1 List of IRES used to construct bicistronic Yellow Fever Viruses IRESType Length Virus (PFU/ml) Mengo Viral 2 569 nt 5 × 10⁵ HCV Viral 3 377nt 2 × 10⁶ Gtx Cellular 227 nt 2 × 10⁵

[0143] To produce the YFV constructs, the complete coding sequencecorresponding to the YFV genome was cloned into a low copy numberbacterial plasmid: pYF-FL. The plasmid can be further manipulated bystandard molecular biology techniques to generate the appropriatemodification in the YFV genome.

[0144] To generate viral genomes containing insertions of foreignsequences, YFV cDNA containing a multiple cloning sites (MCS) ofbicistronic YFV cDNA were constructed by overlap PCR. This procedure wasfollowed by obtaining PCR-amplified IRES-elements containing DNAfragments as shown in Table 1 and the cloning of these fragments intothe pYF-FL plasmid. Additionally, the firefly luciferase or thegreen-fluorescent protein gene was also inserted into the pYF-FLplasmid, 3′ to the different IRES fragments to construct the followingvectors; bYF-M-Luc, bYF-C-Luc, bYF-G-Luc, bYF-M-EGFP, bYF-M-GFP/Zeo. “M”indicates a Mengo IRES; “C” indicates an HCV IRES; and “G” indicates aGtx IRES. The plasmids were then digested at a unique restriction sitepresent at the 3′ end of the genomic cDNA. Subsequent transcription ofthis DNA template by the SP6 RNA polymerase generated a full lengthviral RNA which was transfected in permissive cells in order to obtaininfectious virus. The recombinant viruses are defined in Table 2. TABLE2 Bicistronic recombinant Yellow Fever Viruses. Genome Virus VirusForeign gene Gene Length Length (PFU/ml) YF-17D —   0 nt 10,861 nt 1 ×10⁷ YF-MCS Linker MCS  39 nt 10,900 nt 1 × 10⁷ bYF-M-Luc Luc 1600 nt13,134 nt 5 × 10⁵ bYF-M-EGFP EGFP  726 nt 12,204 nt 2 × 10⁷ bYF-M- GFP +zeocin 1077 nt 12,553 nt 1 × 10⁷ GFP/Zeo

Example 2 Production of Luciferase by Bicistronic YFV Vectors

[0145] Bicistronic YFV vectors containing the luciferase reporter genewere shown to produce luciferase in infected BHK cells. FIG. 2 shows theproduction of luciferase from BHK cells transfected with recombinantbicistronic viruses (bYF-M-Luc, bYF-C-Luc, and bYF-G-Luc) carrying aluciferase reporter gene, along with the appropriate controls, BHK cellstransfected with 17D parental YFV, 17D carrying a Multiple cloning site(MCS) and uninfected cells. Cytoplasmic extracts of the BHK cells wereobtained 24 hours post-transfection. Luciferase activity was measuredfrom the extracts by standard techniques and normalized for proteinconcentration. Transfection of RNA derived from bYF-M-Luc, bYF-C-Luc,and bYF-G-Luc into BHK cells resulted in the production of substantialamount of luciferase activity indicating that the bicistronic YFVrecombinant genomes are capable of expression luciferase. FIG. 3 showsgraphically the time course of luciferase production in BHK cellsfollowing transfection with bYF-M-Luc, bYF-C-Luc, or bYF-G-Luc.

Example 3 Replicative Characteristics of Bicistronic YFV Recombinants

[0146] The replicative characteristics of the bicistronic YFVrecombinants were analyzed by plaque assay and one-step growth curves.The recombinant viruses carrying the luciferase gene or the enhancedgreen-fluorescent-proteins (EGFP) have been shown to replicate at ratesremarkably similar to wild-type parental strain and also achieve nearlyequivalent titers.

[0147] Insertion of the entire luciferase gene (1,500 nucleotides inlength) downstream of the IRES element yielded viable virus thatreplicates with a phenotype similar to the parental virus. To analyzethe phenotype of recombinant virus, BHK cells were infected withrecombinant b-YFV-Luc or parental virus obtained after 1, 2, or 3passages on tissue culture. The BHK cells were incubated at 37° C. for 4days. Plaques produced by bYFV-Luc-recombinant resembled those of theparental virus. In addition, high titer stocks of the recombinant virus(1×10⁶ to 10⁷ PFU/ml) were obtained.

[0148]FIG. 4 also demonstrates the similar replicative characteristicsof the bicistronic YFV recombinants to wild-type viral strains. FIG. 4is a graphical representation of the one-step growth curves of parentalyellow fever 17D strain (YF-17D) and three bicistronic YF recombinantviruses: bYF-M-Luc, and bTF-C-Luc. A similar growth pattern is seen forwild-type YF and all three recombinant viruses in FIG. 4.

[0149] RNA viruses exhibit a high rate of genetic variation (pointmutations, recombination, etc.) which can affect the immunogenicity ofthe vaccine. The genetic stability of the sequences inserted into theYFV genome was carefully examined for possible genetic variations. Toidentify genetic variation, recombinant viral stocks were generated bysequential passages in BHK cells followed by RT-PCR analysis of the YFVgenome. The original genetic structure of the YFV was retained after 3passages in BHK cells, as shown by RT-PCR analysis of BHK cells infectedwith recombinant bYF-M-Luc after three rounds of replication, and BHKcells infected with parental 17D virus. The presence of the foreignsequence was analyzed by RT-PCR of total cytoplasmic RNA from infectedcells.

Example 4 Production of Fluorescence by Bicistronic YFV Recombinants

[0150] Fluorescent bicistronic YFV recombinants were constructed byamplifying full length EGFP or GFP/Zeo fusion (enhanced greenfluorescent protein and green fluorescent protein fused to antibioticzeocin) from pEGFP and pGFP/Zeo (Clontech, CA), and then cloning thesePCR fragments into the NotI and PmeI sites of pbYF-M-Luc by standardtechniques. The constructs containing the EGFP and GFP/Zeo sequenceswere named bYF-M-EGFP and bYF-M-GFP/Zeo, respectively.

[0151] Upon transfection of bYF-M-EGFP or bYF-M-GFP/Zeo viral RNA intoBHK cells, GFP was expressed to high levels in all cells positive forYFV proteins. YFV protein was detected using a mouse hyperimmuneanti-total YFV proteins antibody that had ban conjugated to Alexa 594 (abright, Texas Red-like fluorophore) (Molecular Probes, OR; Alexa 594Protein Labelling Kit). BHK cells infected with bYF-M-EGFP for 48 hourswere fixed and permeablized (CalTag, Ca) in the presence of 0.5 ml Alexa594 fluorescently labeled anti-YFV antibody in a 50 ml total volume.Stained cells were visualized for immunofluorescence using a Leica DMLBmicroscope.

[0152] The percentage of cells positive for both GFP and YFV proteinswas also examined. To determine the percentage of GFP+ virus in a viralstock, bYF-M-EGFP was plaque assayed and GFP+ plaques were visualized,using a long-pass FITC filter, as a green circle of cells. A highpercentage of cells stained positive for both GFP and YFV.

[0153] The viability and stability of recombinant YFV containing SIVsequences were also examined. A modified bYF-M-EGFP containing SIVsequences was shown to maintain the SIV sequences for at least sixpassages by double staining experiments.

[0154] These experiments clearly demonstrate that bicistronic yellowfever viruses are capable of expressing a substantial amount of foreignproteins and retaining the inserted sequences for a large number ofrounds of replication.

Example 5 In Vivo Analyses

[0155] Methods

[0156] Construction of Recombinant Plasmids

[0157] A plasmid encoding the complete YF-17D sequence was used.Bicistronic vectors were constructed by inserting sequences at the 3′noncoding region of the genome. To facilitate cloning, we used a primerextension approach to insert a multiple cloning site (AscI, PacI, PmeI,and SnaBI) exactly 25 nucleotides downstream the end of the yellow feveropen reading frame (ORF). Next, we PCR-amplified IRES-containing DNAfragments and cloned those fragments into pYF-17D. Finally we cloned thefirefly luciferase gene after several different IRES to obtain theplasmids: bYF-Cyr61-Luc, bYF-Gtx-Luc, bYF-HCV-Luc, b-YF-BVDV-Luc,bYF-Mengo-Luc, bYF-Polio-Luc. Constructs bYF-Mengo-eGFP andbYF-Mengo-GFP/Zeo were made by amplification and cloning of full lengtheGFP or GFP/Zeo fusion from peGFP and pGFP-Zeo plasmids (Clontech, PaloAlto, Calif.) respectively, into the NotI and Pmel sites ofbYF-Mengo-Luc plasmid (FIGS. 5A-D).

[0158] DNA Sequencing

[0159] All sequencing reactions were performed on an ABI Prism® 310 DNAGenetic Analyzer (Applied Biosystems, Foster City, Calif.). We appliedMegalign software (DNA Star Inc., Madison, Wis.) for computer-aidedanalysis of sequence data.

[0160] Generation of Viruses from Plasmids.

[0161] In all cases the viral cDNA were digested with Xho I and used astemplates for in-vitro transcriptions driven by the SP6 RNA polymerasein the presence of 7-methyl guanosine cap-structure (mMessage mMachine,Ambion, Austin, Tex.), Transcription products were treated with DNAsefor 15 minutes at 37° C., and precipitated with ethanol/7.5 M ammoniumacetate. The concentration of RNAs was estimated by spectrophotometry.Since XhoI is not a unique site in the bYF-BVDV-Luc andbYF-Mengo-GFP/Zeo constructs, a partial digestion with XhoI was carriedout in order to generate full-length linearized cDNAs. The RNAs weretransfected into BHK-21 cells by electroporation using 2.5 ug of RNA per2×10⁶ cells on an electro cell manipulator 600 (BTX, San Diego, Calif.).

[0162] Cell Lines

[0163] BHK-21 cells were used to make the viral stocks, plaque assay andgrowth curves; and COS were used for the IF experiments.

[0164] Viral Stocks

[0165] Cytopathic effect (CPE) was observed 3 to 5 days followingtransfection. Supernatants of transfected cells were collected after 3days, cleared, aliquoted, titered and stored at −70° C.

[0166] Single-Step Growth Curves

[0167] Subconfluent BHK-21 cell monolayers were washed once with PBS andinfected at a multiplicity of infection (MOI) of 5 PFU/cell. After a 1hr incubation period at 37° C., the cells were washed twice with PBS andthen covered with MEM with Earle's BSS medium supplemented with 10%Fetal Calf Serum. Infected cell cultures were incubated at 37° C. andaliquots were recovered every 12 hours for a period of 3 days. Titerswere determined by plaque assay. Briefly, serial dilutions of viralaliquots were made and added to BHK cell dishes for 1 hour to allowviral adsorption, plates were washed once with PBS before adding 1× MEMand 0.8% agar overlay, plaque assays were then incubated at 37° C. for 5days. Agar overlays were then removed, and plates were stained withvital dye (0.1% crystal violet, 20% ethanol) to reveal viral plaques,which were counted.

[0168] Analysis of Viral RNA by RT-PCR

[0169] After several passages of recombinant viruses on BHK-21 cells,total cytoplasmic RNA was obtained from infected cells using TRIZolfollowing manufacturer's protocol (Life Technologies, Rockville, Md.).Next, reverse transcription was carried out using Superscript II(LifeTechnologies, Rockville, Md.), with random hexamers, 1 ul of cDNAwas added as template in a PCR reaction using rTth (PE Biosystems,Foster City, Calif.) and specific flanking primers for each case.

[0170] Analysis of Viral RNA Replication by Real Time RT-PCR

[0171] HBK cell monolayers were infected with equal concentration ofvirus (MOI=5) and total cellular RNA was extracted at different times toquantified the replication of viral RNA.

[0172] Immunostaining (Immunocyto/Histochemistry)

[0173] Cells were plated on glass cover slips and allowed to attachovernight (1-5×10⁴ cells/cover) (Fisherbrand, Pittsburg, Pa.).Infections were carried out for one hour at 370 C, cells were washedwith PBS before adding media and then placed at 37° C. for 2-3 days.Subsequently, cells were washed with PBS and fixed in 4%paraformaldehyde (PFA) for 10 minutes. After washes incubation withprimary antibodies in PBS buffer containing 0.1% Triton X-100, 2% NCSand 0.02% Sodium azide, Yellow Fever (17D) hyperimmune ascitic fluid(mouse) (National Institute Allergy and Infectious Diseases, Maryland,cat# V525701-562) or IgG fraction of a rabbit anti-Luciferase antiserum(SIGMA, St. Louis, Mo., cat#L-0159) was performed for one hour at roomtemperature. Cells were rinsed three times for 10 minutes with agitationwith PBS and 0.1% Triton X-100 (Bio-Rad Laboratories, Hercules, Calif.).FITC and TRITC-conjugated secondary antibodies (Jackson ImmunOResearchLaboratories, West Grove, Pa.) were incubated for 30 minutes at roomtemperature, protected from light. Finally cells were rinsed three timesfor 10 minutes and mounted in Vectashield medium with DAPI (VectorLaboratories, Burlingame, Calif.).

[0174] Luciferase Assay

[0175] Cell monolayers were washed once with PBS and scraped-off theplate. Extracts were prepared using 100 ul of cell culture lysis reagent(CCLR), (Luciferase Assay System, Promega, Wis.). Ten μl of the extractwere placed in an OptocompI automated luminometer (MGM Instruments,Hamden, Conn.).

[0176] Luciferase Expression in Viral Plaques in Real Time

[0177] After an incubation of 6 days at 37° C., the agar overlay wasremoved and the luciferase substrate (luciferin) was added. Theluciferase activity was measured with a equipment that detect thephotons emitted by the luciferase that is being translated in theinfected cells and is visualized with colors that indicate the level ofluciferase activity. Images of the expression were taken and the plaqueswere observed after developing with crystal violet.

[0178] Bicistronic YF-Mengo-eGFP Stability

[0179] Stock YF-Mengo-eGFP P0 was produced after transfection of BHKcells with in vitro RNA and incubation for 3 days at 37° C. Futurepassages consisted in infection of BHK monolayers at low MOI (MOI=0.01)and incubation for periods of 3 days to generate new viral stocks,namely P1-P5. Plaque phenotype was study by direct observation underfluorescence microscope before the staining with crystal violet. Plaquesexpressing eGFP were identified and the phenotype observed was comparedwith plaques that did not express eGFP. Each viral stock was used toinfect new BHK monolayers and after a 48-hour period the total RNA wasextracted and used as template for RT-PCR to detect the eGFP sequence.

[0180] Expression of eGFP After Cellular Passages Assessed byQuantitative Immunofluorescence

[0181] The same condition was used to infect cells with the differentpassages. The percentage of infected cells that express eGFP wasdetermined for each viral passage. Yellow fever viral proteins weredetected using mouse anti-YFV antiserum and, as a secondary antibody,anti-mouse IG antibody conjugated with TRITC (red fluorescence).

[0182] Processing of Mice Tissues for Cryostat Slides

[0183] Various tissues were extracted from perfused mice to makecryostat slides. The tissues were treated with sucrose 15% (for 3 hours)followed by sucrose 30% (for 1 hours). The tissues were embedded in OCT(Tissue-Tek OCT compound, Sakura, Calif.) for 10 minutes and were frozenin new OCT and kept at −70° C. The slides were made in cryostat, placedin glass charged to favor tissue adhesion and kept at −700 C untilimmunohistochemical staining.

[0184] Immunohistochemistry of Mouse Tissues

[0185] The tissues were rehydrated in PBS for 10 minutes, and then wereplaced in a solution to block unspecific binding of antibodies for onehour at room temperature. The primary antibody was incubated at 4° C.over night. The primary antibodies were: Yellow Fever (YF 17D) or IgGfraction of rabbit anti-luciferase antiserum. The second day the slideswere washed 3 times in PBS for 10 minutes with shaking. The secondaryantibodies were conjugated with FITC, TRITC, or Texas red (JacksonImmunOResearch Laboratories, West Grove, Pa.) and were incubated for 2hours at room temperature, protected from the light. Finally, the slideswere washed 3 times for 10 minutes, and were incubated with a solutioncontaining DAPI before mounting for the microscopic visualization inVectashield medium (Vector Laboratories, Burlingame, Calif.).

[0186] Luciferase Detection In Vivo

[0187] The mice were anesthetized with pentobarbital (70 mg/kg). Asolution of the substrate luciferin (50 mM; 12 mg/Kg., Molecular Probes,Eugene, Oreg.) was injected in the peritoneal cavity 10 minutes beforethe image was taken. The mice were then placed in a dark box. An imagewith fluorescent white light was taken as reference using a photographiccamera with Nikon lenses. A computerized system coupled with the camerawas used to analyze the images.

[0188] The luciferase translated inside the animal produced emission ofphotons that were transmitted through the tissues, collected andintegrated for a period of 10 minutes. An image in colors representingthe light intensity (blue for the less intense and red for the mostintense) was generated by a processor of images Aarhus 20 (Hamamatsu,Bridgewater, N.J.) and were transferred using a module coupled with acomputer with an image processor (Photoshop, Adobe Systems, MountainView, Calif.). The images of reference were superimposed on the colorimages and the annotations were realized using software for graphics.

[0189] Results

[0190] The viral vectors are based on the vaccine strain YF 17D. Therecombinant viruses carry and express large exogenous sequences (2,358nucleotides). The insertions are placed downstream the ORF of the YFVgenome. Several cellular and viral internal ribosome entry sites (IRES)drive the expression of foreign genes. In this report, we describe thedesign and use of a bicistronic vector system into which the IRES of twopicornavirus (Mengo virus (MV) and poliovirus (PV)) a pestivirus (BVDV)and an hepacivirus (HCV), have been inserted into the intercistronicspacer to drive luciferase protein synthesis (FIGS. 5A-D). The sequencesrequired for internal initiation of translation extend at least up tothe authentic viral initiation codon in the case of the Mengo virusIRES, and BVDV IRES, and beyond it in the case of HCV. Thus, internalinitiation of translation results in the synthesis of luciferase proteinwith N-terminal extensions of 7, 4 and 11 amino acids respectively(FIGS. 5A-D). Similarly, although the 3′ boundary of the polio IRES liesupstream of the authentic initiation codon for ease of construction the5′-UTR fragment used extends beyond the authentic viral AUG codon, thuspoliovirus IRES activity results in the synthesis of a luciferaseprotein which has an N-terminal extension of three amino acids (FIGS.5A-D).

[0191] Comparison of IRES Efficiencies in Transfected Cells

[0192] Transfection of in vitro synthesized RNA derived from constructsbYF-Cyr61-Luc, bYF-Gtx-Luc, bYF-HCV-Luc, bYF-BVDV-Luc, bYF-Mengo-Luc,and bYF-Polio-Luc into BHK cells resulted in the production ofsubstantial amounts of the reporter gene, as measured by luciferaseactivity assays. The mengo IRES was the most efficient in drivingdownsteam cistron translation, followed by the BVDV and Polio IRES,which were 7 and 17 fold less efficient than the Mengo IRESrespectively. The cellular IRES (Cyr61 and Gtx), and HCV IRES were muchless efficient in directing internal translation initiation (more than300 fold difference with the Mengo IRES). When luciferase activity wasmeasured 2 and 3 days post-transfection, all constructs showed increasedexpression over time, after this period the virus cause cytopathiceffect to the cells.

[0193] Replication of Bicistronic YFV Recombinants

[0194] In order to assess the efficiency of replication, plaque assayswere carried out, showing that all recombinants carrying the luciferasegene or the enhanced green-fluorescent-proteins (eGFP) generateinfectious viruses in high titers (10⁶ to 10⁷ pfu/ml).

[0195] One Step Grow Curve

[0196] Using the high titer stocks, BHK monolayers were infected andincubated at 37° C., cell suspension samples were taken at differenttime points and were titered by plaque assay. A growth curve was plottedshowing that some viruses (bYF-Gtx-Luc, bYF-Polio-Luc, bYF-Cyr61-LucbYF-HCV-Luc) are produced at low levels at early time points (24 to 48hours post infection) in comparison with the virus YF17D and otherbicistronic viruses (bYF-Mengo-Luc, bYF-BVDV-Luc). After a longerincubation (48 to 72 hours post infection) all the bicistronic virusesreached the level of replication shown for YF-17D.

[0197] Analysis of the Insert Retention by Studying the Replication ofthe Viral RNA in the Infected Cell Utilizing Kinetic RT-PCR

[0198] Although all the recombinant viruses are capable of replicationfrom cell to cell yielding high titers, we do not have information ifthe virus we titer maintained the insert in their genome. To answer thisquestion, we analyzed the replication of the bicistronic genomes in theinfected cell by quantitative amplification. For this reason weextracted RNA at different time points after infection (MOI=5 pfu/cell).The time after infection at which RNA was collected was extended morethan the expected cytopathic effect time for YF17D, because of thepossibility that the recombinant viruses could replicate slowly, and thetime of appearance of cytopathic effect could be modified. The extractedRNA was quantified and used as template for the real time RT-PCR.Specific primers for the YF genome (complementary to the ORF of YFV) andfor the inserted sequence (luciferase) were used. Samples were processedby duplicates for each set of primers, and a constitutively expressedgene (hamster GAPDH) was amplified as internal control to ensure thatthe amount of total RNA used as template was uniform.

[0199] Amplification curves of YF and luciferase were plotted for eachbicistronic YFV. The biistronic virus bYF-gtx-Luc retains the insertduring the period analyzed, which amplification curves for the viralsequence and the luciferase sequence were nearly identical to each otherover the time analyzed. Positive real time RT-PCR was obtained for everybicistronic virus Luc indicating that the insert was maintained duringreplication.

[0200] Yellow Fever Proteins Detected in Cultured Cells byImmunohistochemistry

[0201] BHK and COS cells were infected at low MOI (MOI=0.01 pfu/cell)and processed for immunostaining to detect YF infected cells. After twodays of incubation positive cells for the YF proteins were detected. Todetermine stability of the inserted sequences, bYF-Mengo-Luc was passedon BHK cells several times and then expression of luciferase determinedby immunohistochemistry. About 75% of the infected cells retainedluciferase expression after 5 passages in BHK cells.

[0202] Analysis of the Expression of Luciferase in Infected Cells byDouble Immunohistochemistry

[0203] To evaluate the expression of luciferase by the variousbicistronic viruses, BHK cells were infected with bYF-Mengo-Luc andbYF-Polio-Luc. After 2 days of incubation, the presence of luciferasewas detected by double immunohistochemistry. The presence of YFVproteins was detected using, as primary antibody, mouse YFV-specificantibodies (anti-YF), and, as secondary antibodies, anti-mouse Igconjugated with FITC (green fluorescence). Luciferase was detectedusing, as primary antibody, rabbit luciferase-specific antibodies, and,as secondary antibodies, anti-rabbit Ig antibody conjugated withrhodamine (red fluorescence). Cells expressing yellow fever proteinsexhibit green fluorescence; and cells expressing luciferase exhibit redfluorescence. The results demonstrate that luciferase expression isdetectable in infected cells.

[0204] Quantitative Assessment of the Expression Levels of the 2^(nd)Cistron

[0205] The ability to express luciferase after infection was tested in aquantitative assay. High-titer viral stocks of bicistronic YF-Luciferaserecombinants were produced in BHK cells. To confirm whether therecombinant viruses were infectious and able to express the insertedsequences, BHK cells were infected with the same titer of all viruses,and cell extracts were prepared at different time points after infectionto determine the luciferase activity. Luciferase was readily detectedafter 12 hours post infection and increased exponentially over time upto 48 hours. Bicistronic viruses containing cellular IRES (bYF-Gtx-Luc,and bYF-Cyr61-Luc) and HCV IRES (bYF-HCV-Luc) showed low levels ofexpression, and the rest of the viruses (bYF-Polio-Luc, bYF-Mengo-Luc ybYF-BVDV-Luc) showed at least two fold higher levels. This observationindicates that the YFV genome can accommodate at least 2,358 additionalnucleotides and that the foreign sequences are retained for a number ofpassages.

[0206] Analysis of the Plaque Morphology

[0207] Upon infection of subconfluent BHK cells, YFV and YFV-MCS producecharacteristic large and clear plaques. Conversely, bYF-Mengo-Luc,bYF-BVDV-Luc, bYF-Gtx-Luc, and bYF-HCV-Luc display smaller and blurredplaques. The appearance of scattered big plaques might indicate that theinsert is being lost at different rates in each revertant species.

[0208] In addition, infection with 5-10 p.f.u. per cell of virus (every12 h virus was harvested and titered on BHK-21 cells to obtain a singlestep growth curve) indicated that recombinants replicate at ratesremarkably similar to those of the parental strain 17D, achieving nearlyequivalent titers.

[0209] Analysis of Viral Plaques Expressing Luciferase in Real Time

[0210] After infection with recombinant YF carrying luciferase, BHKcells were overlaid with agar to favor plaque formation. Using equipmentthat allows the detection of light emitted by the cells, we were able tosee the infected cells that express luciferase. After adding thespecific substrate, all the bicistronic viruses that contain luciferasegene showed enzymatic activity and emitted detectable light. Thissuggests that all of the bicistronic viruses express efficiently theenzyme and that the biological activity is maintained.

[0211] The viruses can be divided in two groups, one group of virusesexpresses low levels of luciferase, and includes those containingcellular IRES and the HCV (bYF-Gtx-Luc, bYF-Cyr61-Luc and bYF-HCV-Luc);the second group includes viruses containing viral IRES that expresshigh levels of luciferase (bYF-Polio-Luc, bYF-Mengo-Luc y bYF-BVDV-Luc).Viral plaques are opaque and smaller in size when the virus expressesluciferase. Clear and bigger plaques are indicative of viruses notexpressing luciferase. 93% of bYF-Gtx-Luc virus and 92% of bYF-BVDV-Lucexpress luciferase. It is notable that these two groups of viruses agreewith the ones described before, based on the luciferase activity fromcellular extracts.

[0212] The cells that were infected with highest concentration of virusbYF-Mengo-Luc and bYF-Polio-Luc show a total cytopathic effect at thetime of data collection impairing the visualization of the expression ofluciferase. Whereas the same wells infected with the virusesbYF-BVDV-Luc, bYF-cyr-Luc and bYF-gtx-Luc do not show evidence of totalcytopathic effect after 6 days, and the luminous plaques were clearlyvisible.

[0213] Because the half-life of luciferase is approximately 2 hours, theenzyme detected during the analysis was likely synthesized within a2-hour period before the assay

[0214] In this assay, plaques with different sizes and opacity could beobserved. By comparing plaques obtained by the classic plaque assay(developed with crystal violet), one can distinguish between plaquesthat express high levels of luciferase and plaques that express lowerlevels or do not express luciferase. The vast majority of the plaques ofthe virus bYF-Gtx-Luc (93%) and with the virus bYF-BVDV-Luc (92%)express luciferase.

[0215] Insert Retention in the bYF-Mengo-eGFP Viral Genome

[0216] RNA viruses exhibit a high rate of genetic variation (pointmutations, recombination, etc.). Since this may affect theimmunogenicity of the vaccine we decided to examine the geneticstability of the eGFP sequence inserted into the YFV genome. To thisend, we generated viral stocks by sequential passages in BHK cells withan MOI of 0.01, taking the supernatant after 3 days and analyzing YFVgenome by plaque assay, RT-PCR, and immunocytochemistry.

[0217] All plaques produced by bYF-Mengo-eGFP were small and clear,indicating a slower replication and the presence of the insert.

[0218] By analyzing the plaque morphology we can infer that the insertis lost after the passages, more remarkably after the 3^(rd) passage. Toconfirm this result at the level of a single Yellow Fever infected cell,we checked the amount of cells expressing the EGFP byimmunocytochemistry.

[0219] The presence of eGFP RNA in the bYF-Mengo-eGFP genome wasanalyzed after successive passages in cultivated cells. The results showthat after P5, the presence of RNA genomes containing the eGFP gene isdetectable. However, this technique does not indicate whether the geneis functional, since small mutations or even deletions are not detectedby RT-PCR analysis.

[0220] Bicistronic Virus YF-Mengo-eGFP Stability

[0221] Transfection with in vitro synthesized RNA from plasmidsbYF-Mengo-eGFP and bYF-Mengo-GFP/Zeo resulted in the production ofsubstantial amounts of fluorescence which was easily detected visuallyunder a microscope.

[0222] To determine the percentage of viruses expressing eGFP (eGFP+) inthe viral stocks, bYF-Mengo-eGFP virus was plaqued and cells wereobserved directly under the fluorescence microscope through the plasticby inverting the dish. All the plaques produced by the bicistronic virusbYF-Mengo-eGFP using the stocks P0 to P2 expressed the fluorescentprotein. The passage #P3 showed few bigger plaques that do not expresseGFP, as viewed under the microscope.

[0223] Study of the eGFP Expression After Passages of the Virus bYF-Mengo-eGFP by Quantitative Immunofluorescence

[0224] The stability of the virus bYF-Mengo-eGFP was assessed afterpassages in BHK cells. After passage 3 the virus bYF-Mengo-eGFP isunstable and the percentage of positive cells infected with YF thatexpress eGFP diminished (60% of the infected cells express eGFP). Inpassage 5 only 10% of the infected cells express eGFP. Since theincubation time necessary to observe the positive sign byimmunohistochemistry is reached after 3 days of incubation, at thisperiod all the cells were infected. However, the fluorescenceintensities resulted in variability due to the fact that not all thecells were infected at the same time.

[0225] Selection of Recombinant bYF-Mengo-eGFP With Enhanced InsertRetention Capacity.

[0226] We have selected viruses by FACS sorting eGFP positive infectedcells that are capable to produced bigger plaques and retain theinserted sequences for more rounds of replication. BHK cells wereinfected with bYF-Mengo-GFP and after 24 hours post infection greencells were sorted. At each of round of replication, those cellsexpressing high levels of GFP were selected. Viruses isolated from“green” cells were used to infect BHK cells to obtained a viral stock ofeach passage. The selection procedure was repeated 9 times to obtainviral stock named SP1 to SP45. At 24 hours post infection, SP45 selectedviruses expressed much higher levels of GFP than parental SP1 viruses.Importantly, selected SP45 virus present a larger plaque phenotype,replicate to higher titers and retained the insert for more than 9rounds of replication in tissue culture. These experiments indicate thatrecombinant yellow fever can be isolated that present better replicationand insert retention properties.

[0227] Detection of Yellow Fever Virus After Intracerebral InoculationWith bYF-Mengo-eGFP

[0228] The characterization assays in tissue culture of bicistronicviruses containing the reporter gene luciferase showed that the viruscontaining the IRES derived from Mengo virus belongs to the group ofhigher expression of the reporter gene. For this reason, bYF-Mengo-eGFPwas used in the experiments in mice that are described below. gPVR micesix weeks old were injected IC (intracerebrally) with 10⁵ pfu ofbYF-Mengo-eGFP. Six days post infection the brains were extracted andprocessed for the detection of the presence of the yellow fever virus.

[0229] To detect the presence of genomic RNA containing the insertedsequence, RT-PCR using templates of total RNA from brains was extracted.Virus was extracted from brains and was plated to analyze the phenotyperecovered. To detect the presence of viral proteins and eGFP, cryostatslides were processed for immunohistochemistry.

[0230] RT-PCR From RNA Extracted From Brain of Mice After IntracerebralInoculation With the virus bYF-Mengo-eGFP

[0231] To evaluate the presence of viral RNA as well as the retention ofthe insert in the genome, RNA was extracted from brains of mice infectedintracerebrally with the bicistronic viruses. We performed RT-PCR todetect the presence of viral RNA in the brain samples. The RT-PCR for YFshows that both the genome of the virus YF 17D and the bicistronicbYF-Mengo-eGFP replicate in the nervous tissue, and viral genomes weredetectable 6 day after the IC injection. The RT-PCR for eGFP shows thatviral RNA that contains the insert sequence of eGFP is detectable 6 daysafter the IC injection of mice with the virus bYF-Mengo-eGFP. A fragmentthat corresponds to the actin gene was apmplified as control that isfound in all RNA used as templates from the brain samples (either withor without infections). A fragment of the virusYF 17D present in boththe parental strain and in the construct bYF-Mengo-eGFP was alsoamplified. In both controls the results were as expected.

[0232] Immunohistochemistry of Mouse Tissue

[0233] To study the presence of viral proteins in the brains of infectedmice, we performed immunohistochemistry on brains extracted afterinjection with the viruses YF17D and bYF-Mengo-eGFP. Brains fromuninfected mice were processed as controls. The brains were extracted atdifferent time points after the IC inoculation using different viralstocks and different amount of virus. The cells infected with the YFVwere detected with specific red fluorescence in the slides of brainsobtained after the infection with the parental virus YF17D and withrecombinant virus bYF-Mengo-eGFP. The slides were observed under thefluorescent microscope to detect the expression of eGFP. Direct greenflorescence was not detected in the processed tissues. Extractions weremade at different days post-injection.

[0234] In every case, eGFP expression was detected. To explore thepossibility that the levels of expression of eGFP were insufficient todetect the direct fluorescence under the microscope, specific anti-GFPantibodies were used. The expression of eGFP was not detected, either bydirect fluorescence or by immunohistochemistry using specificantibodies. For a complete inspection cryostat slides were made in thecoronal plane and in the horizontal plane y horizontal. The absence ofsignal for eGFP suggested that the construct bYF-Mengo-eGFP does notexpress the second cistron in a stable way. This observation agrees withthe plaque assay using the virus recovered from brains of mice infectedwith the same virus bYF-Mengo-eGFP, which does not express eGFP andshows the same size as the plaque-generated with the parental YF17D.

[0235] Assessment of Luciferase Expression in Mice Tissues

[0236] Six week old BALB/C mice were injected IC with the bicistronicvirus: bYF-Polio-Luc, bYF-Mengo-Luc and bYF-BVDV-Luc. As negativecontrols, mice were injected with YF17D and PBS. After 2, 3 or 6 dayspost-infection brain homogenates were made and the luciferase activitywas measured. In the first attempt, whole brain extracts were made inwhich no luciferase activity was detected. One of the explanations isthat the enzyme activity it is diluted in the extract of the wholebrain. It may be necessary to obtain homogenates from regions close tothe IC inoculation site of the virus to detect luciferase activity, sothat the enzyme is concentrated in the site of the inoculation. To avoidthe dilution of the expressed protein, four sections of the infectedbrains were processed independently. All the bicistronic viruses showedluciferase activity in the extracts of brains at different days afterthe IC inoculation. Significant levels of luciferase were detected inthe brains of mice injected via IC after 2, 3 and 6 days after theinfection with the bYF-BVDV-Luc; after 2 and 3 days with the virusbYF-Polio-Luc, and only after 2 days with the virus bYF-Mengo-Luc.

[0237] Detection of Luciferase Expression in Live Mice

[0238] Utilizing a non-invasive method, it is possible to detect theenzymatic expression of luciferase in live mice. The light it istransmitted trough the tissues, and bioluminescence is used to monitor(externally and in a quantitative manner) the expression of theluciferase enzyme in mice. This is a sensitive and quantitative modelthat provides an analysis in real time of the expression of the enzymeand facilitates monitoring of individual animals. Using this technology,without the need to sacrifice the mice, the bicistronic viruses thatcontain the luciferase gene were injected into mice to study theactivity of luciferase in vivo. The light produced by the infected cellsis transmitted through the anesthetized mice. In this experiment, 20mice were injected via IV, IP and IC. Images were taken after theinjections with each route of inoculation, the IC inoculation showedhigh levels of expression of luciferase when using the virusbYF-polio-Luc (6.6×10⁵ total counts).

[0239] Based on the previous results, we used IV inoculation for newexperiments with the objective to follow the kinetics of expression ofthe second cistron. The results showed that in 3 out of 5 mice infectedwith the virus bicistronic bYF-Polio-Luc, the maximum expression isreached at 5 days after the injection and the amount of signal comesfrom the hepatic region. Similar results were obtained in 2 out of 3mice injected with the virus bYF-Mengo-Luc and in 1 out of 5 miceinfected with the virus bYF-BVDV-Luc. In the mice used as controls, nosignificant expression of luciferase was detected.

[0240] All publications and patents cited in this specification areherein incorporated by reference as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. The citation of any publication is for its disclosureprior to the filing date and should not be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention.

[0241] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A live, replication-competent, recombinantbicistronic flavivirus comprising, in order from 5′ to 3′, a 5′untranslated region (UTR) from a parent flavivirus, an open readingframe (ORF) encoding viral proteins of the parent flavivirus, aninternal ribosome entry site (IRES), an exogenous nucleic acid moleculethat encodes a polypeptide other than a polypeptide encoded by theparent flavivirus, and a 3′ UTR from the parent flavivirus.
 2. Therecombinant flavivirus of claim 1, wherein said recombinant flavivirusis attenuated.
 3. The recombinant flavivirus of claim 1, wherein saidparent flavivirus is yellow fever virus.
 4. A live,replication-competent, attenuated yellow fever virus (YFV), comprising,in order from 5′ to 3′, a 5′ untranslated region (UTR) from a parentYFV, an open reading frame (ORF) encoding all viral proteins of theparent YFV, an internal ribosome entry site (IRES), an exogenous nucleicacid molecule that encodes a polypeptide other than a polypeptideencoded by the parent YFV, and a 3′ UTR from the parent YFV.
 5. Therecombinant flavivirus of any one of claims 1-4, wherein infection of ahost cell with the recombinant flavivirus provides for expression of theexogenous nucleic acid in the host cell and production of thepolypeptide encoded by the exogenous nucleic acid.
 6. The recombinantflavivirus of any one of claims 1-4, wherein the exogenous nucleic acidcomprises a nucleotide sequence that encodes a polypeptide from apathogenic agent other than the parent flavivirus.
 7. The recombinantflavivirus of any one of claims 1-4, wherein the exogenous nucleic acidcomprises a nucteotide sequence that encodes a tumor antigen.
 8. Therecombinant flavivirus of any one of claims 1-4, wherein the exogenousnucleic acid comprises a nucleotide sequence that encodes a therapeuticpolypeptide.
 9. A method of delivering a polypeptide to a host, themethod comprising introducing a recombinant bicistronic flavivirus ofclaim 1 into a host that is susceptible to infection with a flavivirus,wherein infection of a host cell with the recombinant flavivirusprovides for expression of the exogenous nucleic acid in the host celland production of the polypeptide encoded by the exogenous nucleic acid.10. The method of claim 9, wherein the polypeptide is associated with amicrobial pathogen other than the parent flavivirus.
 11. The method ofclaim 9, wherein the polypeptide is a tumor antigen.
 12. The method ofclaim 9, wherein the polypeptide has therapeutic activity.
 13. Themethod of claim 9, wherein the polypeptide is exported to the host cellsurface.
 14. The method of claim 13, wherein the polypeptide ispresented on the surface of the host cell with a majorhistocompatibility antigen.
 15. The method of claim 9, wherein thepolypeptide is secreted from the cell.
 16. The method of claim 9,wherein the polypeptide remains in the cytoplasm of the cell.
 17. Apharmaceutical composition comprising a recombinant flavivirus of anyone of claims 1-4; and a pharmaceutically acceptable excipient.
 18. Amethod for eliciting an immune response to an antigen in a mammaliansubject, the method comprising administering a recombinant flavivirus ofclaim 1 or claim 4 to the mammalian subject, wherein the recombinantflavirus encodes a polypeptide antigen, and wherein infection of a cellof the subject with the recombinant flavivirus provides for expressionof the exogenous nucleic acid in the cell and production of thepolypeptide encoded by the exogenous nucleic acid.
 19. The method ofclaim 18, wherein the antigen is a host antigen.
 20. The method of claim18, wherein the antigen is an antigen associated with a pathogenicmicroorganism other than the parent flavivirus.
 21. A method ofpreventing tumor growth, the method comprising administering arecombinant flavivirus of claim 1 or claim 4 to a mammalian subject notbearing a tumor, wherein the recombinant flavivirus comprises atumor-associated antigen/epitope-encoding nucleic acid, such that therecombinant flavivirus enters a cell of the mammalian subject, thetumor-associated antigen is expressed on the host cell surface and/orpresented in the context of an MHC molecule (e.g., MHC Class I), and animmune response is elicited to the tumor-associated antigen.
 22. Amethod of producing an exogenous polypeptide, the method comprisingintroducing a recombinant bicistronic flavivirus of any one of claims1-4 into a susceptible host cell under conditions that favor productionof the exogenous polypeptide by the host cell.
 23. The method of claim22, further comprising purifying the exogenous polypeptide.